Adapting Hydralazine as a Reliable Low-Toxicity Alternative to Cobalt Chloride for Hypoxia Modeling in Breast Cancer Cells

Background: Hypoxia represents a critical characteristic of the breast cancer microenvironment, responsible for tumor progression and therapeutic resistance. Although cobalt chloride (CoCl₂), which is used to induce cellular hypoxia, its application is restricted by severe cytotoxicity and off-target side effects. Objective: To evaluate hydralazine as an alternative for hypoxia induction in human MCF-7 breast cancer cells. Methods: MCF-7 cells were treated with increasing concentrations of hydralazine and CoCl₂ to compare the efficacy and cytotoxicity. Cell viability and stabilization of hypoxia-inducing factor-1 alpha (HIF-1α) were assessed using MTT and Western blotting assays, respectively. GAPDH protein was used as a loading control. Results: Hydralazine maintained near 98% cell viability across all tested doses up to 1600 µM at 24 hours, whereas CoCl₂ treatment resulted in dose-dependent cytotoxicity with a significant viability reduction to approximately 50% at the same maximum concentration. Furthermore, prolonged exposure (72 hours) to 1600 µM CoCl₂ caused severe cytotoxicity (